1. Field of the Invention
The present invention relates generally to virus-like particles (VLPs) for use in delivering a polypeptide of interest to a subject and to nucleic acid molecules encoding same. In particular, the present invention takes advantage of features of avian hepadnavirus particles to generate stable recombinant VLPs which are useful in the delivery to a subject of a polypeptide of interest. The polypeptide of interest may comprise one or more antigens capable of eliciting an immune response. The VLPs comprising the polypeptide of interest are useful for delivery of an antigen to the immune system of a subject or in the presentation of epitopes for the detection of antibodies in in vitro assays. The present invention extends, inter alia, to plasmids, cells, kits and methods which are useful in the generation, production, delivery and monitoring of the instant VLPs.
2. Description of the Prior Art
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
Reference herein to prior art, including any one or more prior art documents, is not to be taken as an acknowledgment, or suggestion, that said prior art is part of the state of the art.
The hepadnaviruses are a family of enveloped DNA viruses. Assembly of mammalian hepadnaviruses, such as hepatitis B virus, is complex and mature virions are formed by the interaction of preformed cytoplasmic core particles with pre-assembled surface proteins on the host endoplasmic reticulum (ER) membrane. Following interaction with appropriate portions of envelope proteins, the nucleocapsids bud into the lumen of the ER along with a 1000-fold excess of empty, subviral particles (SVPs) and assembly is completed in an intermediate, pre-Golgi compartment (as reviewed by Nassal, M., Curr. Top. Microbiol. Immunol., 214:297-337, 1996).
In many studies, virus-like particles (VLPs), which lack nucleocapsids, have proven to be promising candidate vaccines since they: (i) are non-infectious and therefore safe to produce and use, (ii) are more immunogenic than subunit vaccines because they provide the necessary spatial structure for display of epitopes, and (iii) elicit humoral, cell-mediated and importantly, mucosal immunity (Krueger, et al., Biol. Chem. 380:275-276, 1999).
A recent example of a successful VLP vaccine, currently in clinical trials, is the recombinant papillomavirus major capsid protein (L1) VLP, which prevents infection by inducing a strong neutralizing antibody response (Frazer, Virus Research, 89:271-274, 2002).
The hepatitis B virus (HBV) subviral particle (HBsAg-S) has been viewed as a candidate to produce recombinant VLPs. Several studies have examined which domains are suitable for insertion of foreign epitopes (Bruss et al., EMBO J., 13:2273-2279, 1994., 1994; Delpeyroux et al, J. Mol. Biol. 195:343-350,1987), including N terminal fusion of the preS domain (Prange, et al, J. Gen. Virol. 76:2131-2140, 1995).
Most recently, particles carrying small, 35 amino acid insertions of the hepatitis C virus (HCV) hypervariable region 1 of the E2 envelope protein into the exposed ‘a’ determinant in the second hydrophilic loop have successfully elicited antibody responses (Netter, et al, J. Virol. 75:2130-2141, 2001). Notably, there have been limitations to the size of the inserts tolerated for particle stability and a loss of immune reactivity to the ‘a’ determinant of HBsAg when particles were produced in a mammalian cell system (Prange et al, 1995, supra; Bruss et al, J. Virol. 65:3813-3820, 1991).
Particle instability with large fusions has recently been overcome with a Dengue virus/HBsAg fusion by expression in yeast (Bisht, et al, J. Biotechnology. 99:97-110, 2002).
However, in all these cases, in order to assemble chimeric particles, the recombinant S protein must assemble with wild type S subunits. These extended S chains present a difficulty for inclusion in the tight envelope lattice formed by the HBsAg (which excludes L) and so their number is limited, and consequently the immune response generated to the foreign epitopes is low.
Accordingly, there is a need for improved VLPs which efficiently incorporate polypeptides of interest.